Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that causes acute, hospital-acquired infections as well as chronic disease in patients with cystic fibrosis. Though the organism is ubiquitous in the environment and possesses many virulence factors found in other gram-negative bacteria, it rarely causes disease in healthy hosts. Acute infection by P. aeruginosa usually occurs in the setting of pre-existing epithelial tissue damage; this is mirrored by increased bacterial adherence, invasion and cytotoxicity toward damaged epithelial monolayers in vitro. This grant proposal describes experiments to elucidate the molecular basis for P. aeruginosa interactions with epithelial cells. Its primary objective is to understand how pathogen-host cell interactions result in the activation of contact-dependent virulence factors such as the Type III secretion system. We have defined a novel set of P. aeruginosa mutants that can no longer interact with epithelial cells to promote internalization. The ability of these mutants to cause in vitro cytotoxicity will be directly assayed in Specific Aim #1, allowing us to identify a subset of mutants deficient for interactions required for Type III mediated virulence. These mutants will be tested in a murine model of acute pneumonia to confirm that these mutations also result in attenuated virulence toward the intact host. We also present preliminary data about two mutants, ide-2 and ide-9, which demonstrates that we have already identified two putative P. aeruginosa proteins required for epithelial cell interactions leading to Type III dependent virulence. Specific Aims #2 and #3 discuss in detail the characterization of these two mutants. The characterization of bacterial molecules and signaling pathways underlying host cell recognition and contact-dependent virulence in P. aeruginosa will provide novel targets for treating and preventing Pseudomonas colonization and disease. Such insights are likely to prove applicable to other gram-negative pathogens of animals and plants, which similarly activate Type III secretion of bacterial effectors required for virulence in response to poorly understood signals that follow host cell contact. [unreadable] [unreadable]